The use of proteins, peptides and polynucleotides such as DNA and RNA (including small interference (si) RNA) in therapy or in preventive medicine is limited because they are generally impermeable through various biological barriers (e.g., blood-brain barrier (BBB) and membrane barriers of the circulatory system, intestinal track, skin and lungs) and sensitive to proteolytic enzymes, thus not surviving the passage from the site of administration to the site of action. These limitations result in poor pharmacokinetics (PK), preventing or limiting their use in the treatment of neurological diseases and in diseases in other organs of the body.
Many drugs and biologically active molecules cannot penetrate the BBB and thus require direct administration into the CNS tissue or the cerebral spinal fluid (CSF) in order to achieve a biological or therapeutic effect. Even direct administration into a particular CNS site is often limited due to poor diffusion of the active agent because of local absorption/adsorption into the CNS matrix. Present modalities for drug delivery through the BBB entail disruption of the BBB by, for example, osmotic means (hyperosmotic solutions) or biochemical means (e.g., use of vasoactive substances such as. bradykinin), processes with serious side effects.
In order to fulfill the therapeutic potential of peptides, proteins and nucleotides and other agents with poor PK, a non invasive delivery method is required that will distribute the agent at the desired area of the target site (e.g., a wide area of an organ such as the brain), will have good blood circulatory lifetime for the delivery platform, will penetrate through biological barriers and will have a selective disruption mechanism.
Small interference RNAs (siRNAs) are an example for polynucleotides which would have a highly promising therapeutic potential if only their PK could be improved. RNA interference is a powerful strategy to inhibit gene expression through specific mRNA degradation mediated by siRNAs. However, in vivo application of siRNAs is severely limited by their instability and poor delivery to target cells and target tissues. siRNAs could be an alternative therapy of glioblastoma, a brain tumor highly resistant to chemotherapy and radiotherapy. Gene silencing is a promising approach for inhibiting the proliferation of this type of tumor and several target genes may be considered for this therapeutic strategy, such as epidermal growth factor receptor variant III, which is expressed in 40-50% of gliomas, and the phosphoinositide 3-kinase (PI3K)/Akt pathway, which plays a crucial role in medulloblastoma biology. Targeting of such oncogenic pathways can be achieved by gene silencing with RNA interference. However, before RNA interference can be exploited for brain tumor therapy, several obstacles have to be overcome, such as the instability of siRNAs in the blood stream and their impermeability through the BBB.
An efficient delivery system for proteins, peptides, polynucleotides and other biologically active agents should protect the agents while they are being transported, allow them to pass intact through biological barriers such as the BBB, and target them to the site of action by a mechanism that releases them specifically at that site. In order to achieve such performance, such a delivery system should preferably comprise nano-sized drug carriers which are stable in biological fluids, penetrate intact various biological membranes and have a selective disruption mechanism. In addition, such a carrier should be able to encapsulate significant amounts of the active agent whereby many molecules per vesicle or carrier are targeted to a particular site or organ. There are, however, no currently efficient delivery systems wherein all these necessary properties are combined within one delivery system.
Complexation of the anionic carboxyfluorescein (CF) with single headed amphiphiles of opposite charge in cationic vesicles, formed by mixing single-tailed cationic and anionic surfactants has been reported (Danoff et al. 2007). Wang et al. (2006) disclose complexation of the anionic CF with bilayered vesicles formed from cetyl trimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS). The CTAT-rich (cationic) vesicles were shown to capture the CF with high efficiency (22%). The ability of these vesicles to capture and hold dyes is very high (>20%) when the excess charge of the vesicle bilayer is opposite to that of the solute (i.e., CTAT-rich vesicles capture anionic solutes very efficiently, whereas SDBS-rich vesicles efficiently capture cationic solutes).
U.S. Pat. No. 6,358,523 discloses macromolecule-lipid complexes, macromolecule targeting and delivery to various biological systems.
WO 02/055011 and WO 03/047499, both of the same applicant, disclose amphiphilic derivatives composed of at least one fatty acid chain derived from natural vegetable oils such as vernonia oil, lesquerella oil and castor oil, in which functional groups such as epoxy, hydroxy and double bonds were modified into polar and ionic headgroups. The amphiphiles of WO 02/055011 and WO 03/047499 comprise one or more ionic or polar headgroups and at least one hydrogen-bonding group located either within said headgroup and/or in close proximity thereto. These amphiphiles are capable of spontaneously forming vesicles and micelles owing to their polar and ionic headgroups.
WO 03/047499 discloses bolaamphiphiles (vesicle-forming amphiphilic compounds bearing two headgroups), having at least one headgroup containing a selectively cleavable group or moiety such as a residue of a choline or phenylalanine derivative. The cleavable group or moiety is cleaved and the vesicles disrupt and release their load under selective conditions, which include change of chemical, physical or biological environment. These vesicles are preferably cleaved enzymatically in a biological environment such as the brain or the blood. The vesicles or liposomes made from these amphiphilic compounds are highly stable, beyond what is achievable with the lipids and surfactants used in the current state of the art, and suitable for delivery of a therapeutic substance or a diagnostic agent specifically to a target organ or tissue.
The prior art does not emphasize the benefits of using multi-headed amphiphiles for targeted delivery. Simultaneous complexation and encapsulation of small molecules and macromolecules such as peptides, proteins and nucleotides within vesicles of bolaamphiphiles or multi-headed amphiphiles bearing selectively cleavable groups, is not disclosed in the prior art either. However, it is the use of such multi-headed amphiphiles and particularly bolaamphiphiles that can achieve the desired combination.